One important function of the human hematopoietic system is erythropoiesis, which is the production of red blood cells or “erythrocytes.” Erythropoiesis is vitally important as it is the process whereby new red blood cells are generated as old red blood cells degenerate. In this way, a continuous supply of red blood cells is ensured, thereby guaranteeing the continuous oxygenation of biological tissues.
In humans, erythropoiesis occurs in the presence of a protein hormone called “erythropoietin” abbreviated as “EPO” which serves to stimulate the division and differentiation of progenitor cells located in bone marrow. Over a period of about four days, progenitor cells mature into red blood cells and are released into the general circulation. A typical red blood cell lives for about four months in the systemic circulation. Typically, control of the erythropoietic cycle is governed by a negative feedback mechanism whereby the increased oxygenation of the tissues caused by increased numbers of red blood cells results in a decrease in the production of erythropoietin.
Naturally-occurring EPO is a glycoprotein hormone with 165 amino acids, 4 glycosylation sites on amino-acid positions 24, 38, 83, and 126, and a molecular weight of about 34,000. EPO is produced in vivo as a precursor protein with a signal peptide of 23 amino acids and can occur in three forms. The DNA sequences encoding EPO have been reported. See, for example, U.S. Pat. No. 4,703,008.
Pharmacologically, EPO has been administered to humans for the treatment of a variety of conditions, including the treatment of patients suffering from anemia. Specifically, for example, EPO is indicated for (a) anemia associated with chronic renal failure, (b) anemia related to zidovudine therapy in HIV-infected patients, and (c) anemia in cancer patients undergoing chemotherapy. In addition, EPO has been used to reduce allgeneic blood transfusion in patients undergoing surgery, and for pruritus associated with renal failure.
One drawback associated with current forms of EPO therapy is the frequency of dosing. Because EPO therapy typically requires daily injections, patients dislike the inconvenience and discomfort associated with this regimen.
One proposed solution to these problems has been to provide a prolonged release form of EPO. For example, U.S. Pat. No. 5,416,071 describes a water-soluble composition comprising EPO, hyaluronic acid, and human serum albumin. Such compositions, however, may not provide sufficient activity over the desired period of time.
Others have suggested the use of PEGylation technology, or the attachment of a poly(ethylene glycol) derivative to a protein such as EPO, in order to prolong EPO's in vivo half-life. For example, U.S. Patent Application Publication No. 2004/0082765 describes conjugation of PEG derivatives bearing a succinimidyl lower fatty acid ester (such as a PEG derivative bearing succinimidyl propionate or succinimidyl butyrate moiety).
U.S. Patent Application Publication No. 2002/0081734 describes preparing a mutein of EPO having a cysteine residue introduced at the thirty-eighth position and then PEGylating the mutein at the introduced cysteine residue via a PEG-maleimide derivative. No specific structure of the PEG-maleimide derivative was described in the publication.
U.S. Pat. No. 6,753,165 describes methods for making soluble proteins (including EPO) having free cysteines. The publication also describes modifying the soluble proteins by attaching a PEG moiety at the free cysteine via a PEG derivative bearing a vinylsulfone, maleimide or iodacetyl moiety. With respect to attaching the PEG, the publication describes the necessity for performing a partial reduction step in order to increase the relatively unreactive free cysteine on the protein. International Patent Publication WO 90/12874 also describes cysteine-added variants of EPO, wherein a PEG is attached to the added cysteine residue.
International Patent Publication WO 01/76639 describes myelopoietin conjugates, which refers to a fusion protein prepared from a modified human IL-3 polypeptide sequence linked to another molecule such as EPO.
U.S. Pat. No. 6,077,939 describes a process for attaching a PEG to the N-terminal alpha carbon of a protein (such as EPO) that has previously been subjected to a transamination reaction. The described conjugates can contain a PEG linked to EPO via a hydrazone, reduced hydrazone, oxime, or reduced oxime linkage.
U.S. Pat. No. 6,340,742 describes PEG-EPO conjugates wherein EPO is modified by the addition of from 1 to 6 glycosylation sites and covalently linked to from one to three lower alkoxy poly(ethylene glycol) groups, each poly(ethylene glycol) group attached via a specific linkage. The described conjugates are prepared by first “activitating” the EPO by covalently linking one or more protected thiol groups to EPO, followed by removal of the group protecting the thiol. Once the protecting group is removed, the step of attaching certain reagents at the unprotected thiol is performed.
U.S. Patent Application Publication No. 2003/0191291 describes proteins having EPO activity that are prepared using non-recombinant technology. The publication further describes such proteins that are polymer-modified in a defined manner.
U.S. Patent Application Publication No. 2002/0115833 describes an EPO glycoprotein covalently linked to one poly(ethylene glycol) group by way of a specific linkage containing an amide bond with the N-terminal alpha-amino group of the EPO glycoprotein.
EP 0 714 402 describes conjugates of polymers with proteins such as EPO. The poly(ethylene glycol) polymers used to form the conjugates have molecular weights of up to about 10,000.
International Patent Publication WO 96/40792 describes conjugates prepared from a polymer comprising the following structure: Poly(—O—C═O—Y)m, wherein Y is a halogen or nitrile, m is an integer from 1 to 25, and Poly defines a synthetic or a naturally occurring polymer.
Notwithstanding these described conjugates, however, there remains a need to provide conjugates or compositions of EPO that satisfy one or more of the following: conjugates formed from different PEG derivatives (e.g., PEGs having different structures, reactive groups, and so forth); conjugates formed from PEG derivatives having different weight average molecular weights (e.g., greater than about 10,000); conjugates wherein the polymer is not attached primarily at lysine position 52; conjugates formed from recombinant EPO rather than muteins or fusion proteins thereof; compositions that are substantially homogeneous in terms of their EPO-PEGmer content (e.g., monoPEGylated EPO, diPEGylated EPO, etc.), compositions that are well-defined and reproducible in terms of EPO-PEGmer content and positional isoforms thereof, and conjugates that can be formed in relatively few steps and without the need to carry out partial reduction steps or other synthetic transformations (e.g., transamination reactions, addition of thiol groups, and so forth). Ideally, such a conjugate will possess suitable bioactivity in vivo, and possess a circulating half-life such that blood or plasma levels of EPO are sustained over a longer period of time in comparison to currently marketed formulations—such that a pharmaceutical preparation comprising such a conjugate can be administered less frequently than the currently marketed formulations, thereby providing a distinct advantage over currently available EPO formulations.
Thus, there remains a need in the art to provide additional, beneficial conjugates of water-soluble polymers and moieties having EPO activity. Among other things, one or more embodiments of the present invention is therefore directed to such conjugates as well as to compositions comprising the conjugates and related methods as described herein, which are believed to be new and completely unsuggested by the art.